Abstract The obesity pandemic brings with it multiple attendant metabolic comorbidities, including Type 2 Diabetes (T2D) and Non-Alcoholic Fatty Liver Disease (NAFLD). Both T2D and NAFLD are inadequately treated with currently available therapy; although multiple medications are approved for T2D, few address the underlying problem ? insulin resistance. In addition, no medications are approved for NAFLD, the leading cause of chronic liver disease and fastest-growing reason for liver transplantation. Clearly, a wider net for potential therapeutics must be cast in order to stem the tide of obesity-related illness. Notch is a highly conserved family of proteins critical for cell fate decision-making, but less is known about Notch action in mature tissue. We have shown that Notch signaling is present at low levels in normal liver, but increased markedly in livers from diet-induced or genetic mouse models of obesity, and similarly in obese patients with T2D or NAFLD. Increased Notch signaling was found in hepatocytes and immune cells, but not other nonparenchymal liver cells. Next, to test causality, we generated mice lacking hepatocyte Notch signaling ? these mice, when challenged with high-fat diet feeding, showed improved glucose tolerance and a parallel decrease in hepatic steatosis. Conversely, mice with constitutive hepatocyte Notch activity showed glucose intolerance and fatty liver even when fed normal chow diet. We observed similar effects in Notch loss- and gain-of-function mice fed a novel fibrosis-provoking diet. Here, we will examine the mechanisms underlying activation of hepatic Notch signaling, and downstream effectors of this maladaptive response, with ultimate objective to ameliorate obesity-induced metabolic complications. In Aim 1, we will determine if increased Jag1 and Notch1 expression in signal-sending and -receiving hepatocytes, potentially due to parallel increase in Fto and FoxO1 activity in the insulin-resistant liver, coordinate increased liver Notch activity and downstream pathology in obesity. In Aim 2, we test how Notch activity leads to fatty liver, with the hypothesis that Notch- induced PHLPP2 degradation allows unchecked insulin action and persistent de novo lipogenesis. Finally, in Aim 3, we elucidate mechanism and repercussions of the additional increased Notch activity in hepatic macrophages in obesity. Achieving the goals of this application will identify the underlying mechanism of increased liver Notch signaling in obesity, delineate mechanistic determinants of Notch-induced lipogenesis, and may uncover the potential use of Notch inhibitors for treatment of T2D and NAFLD.